Matrix-assisted laser desorption/ionization mass spectrometry analysis and thiol-group determination of isoforms of bovine cytochrome c oxidase, a hydrophobic multisubunit membrane protein.

Matrix-assisted laser desorption/ionization-mass spectra (MALDI-MS) are obtained from entire bovine heart (H) and liver (L) cytochrome c oxidase membrane protein complexes. Molecular masses of most of the subunits are in excellent agreement with the published sequences. Some corrections are necessary for the nuclear coded subunit IX, which is N-acetylated, and X, with a corrected C-terminal peptide sequence. The mass values of two of the three tissue-specific subunits (VIII-L and X-L) are not in agreement with the DNA-deduced sequences and have been corrected by protein sequencing. For the investigation of the cysteine status 7-diethyl-amino-3-(4'-maleimidylphenyl)-4-methylcoumarin proved to be an excellent site-specific reagent. MALDI-MS with the SH-reacted enzyme indicates disulfide bridges only in subunit VII and a distorted tetrahedral S coordination of the zinc in subunit VI.

Two novel isoneolacto-undecaglycosylceramides carrying Galalpha1-->3Lewis(x) on the 6-linked antenna and N-acetylneuraminic acidalpha2-->3 or Galactose alpha1-->3 on the 3-linked antenna, expressed in porcine kidney.

Three sialosylated and three neutral glycosphingolipids sharing a common iso-neolacto core were isolated from porcine kidney cortex. They were purified by preparative HPTLC, and were characterized by partial exoglycosidase hydrolysis followed by thin layer chromatography and immunostaining with anti-Galalpha1-->3Gal, anti-type 2 lactosamine and anti-Lewis(x) antibodies, methylation analysis, MALDI-TOF mass spectrometry and 1H-NMR spectroscopy. Among neutral glycolipids, one was a known structure, VI3VI'3(alphaGal)2-iso-nLc8Cer, and two were novel structures differing by the number of Galalpha3Lewis(x) determinants: VI3VI'3(alphaGal)2V'3alphaFuc-iso-nLc8, and VI3VI'3(alphaGal)2 V3V'3(alphaFuc)2-iso-nLc8. The single Galalpha3Lewis(x) determinant was found on the 6-linked antenna. Among sialosylated glycolipids, two had been previously found in other species and tissues, VI3VI'3(NeuAc)2-iso-nLc8, and VI3NeuAcVI'3alphaGal-iso-nLc8. A novel structure was discovered presenting a Galalpha3Lewis(x) determinant on the 6-linked antenna and a N-acetylneuraminic acid on the 3-linked antenna, VI3NeuAcVI'3alphaGalV'3alphaFuc-iso-nLc8. These results indicate that, in vivo, the porcine kidney alpha3fucosyltransferase synthesizes the Gala3Lewis(x) determinant, acting on the 6-linked before the 3-linked Galalpha3neolactosamine, and appears unable to synthesize the sialosylated Lewis(x) determinant on neolactoseries glycolipids.

Accurate determination of the molecular weight of the major surface layer protein isolated from Clostridium thermosaccharolyticum by time-of-flight mass spectrometry.

Matrix-assisted laser desorption with concomitant ionization, in combination with a linear time-of-flight mass spectrometer, was used to analyze underivatized and hard-to-solubilize surface layer proteins and glycoproteins by depositing them on top of a microcrystalline layer of the matrix alpha-cyano-4-hydroxycinnamic acid. Use of this special sample preparation technique allowed the first successful desorption-ionization of intact surface layer proteins and accurate determination of their molecular weights by mass spectrometry. The molecular mass of the monomeric subunit of the major surface layer protein isolated from Clostridium thermosaccharolyticum E207-71 was determined to be 75,621 +/- 81 Da. The obtainable mass accuracy of the technique is conservatively considered to be within +/- 0.2%. This result deviates from that given by sodium dodecyl sulfate-polyacrylamide gel electrophoresis by approximately 7.4 kDa because this method is strongly affected and biased by the three-dimensional structure of this type of surface protein. With the apparent advantages of unsurpassed mass accuracy, low dependence on the physicochemical properties of the surface layer proteins, and high sensitivity, it can be concluded that a linear time-of-flight instrument combined with UV matrix-assisted laser desorption with concomitant ionization is better suited for molecular weight determination than is gel electrophoresis.